MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Mutations in the X-linked human MECP2 gene cause of Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To understand the molecular mechanism of RTT, it is important to study how MeCP2 dynamically regulates gene transcription, and to reveal the physiological significance of such regulation. Recent biochemical analysis has identified 8 phosphorylation sites on the MeCP2 protein. Among these, serine 80 (S80) is phosphorylated in resting neurons but dephosphorylated in active neurons, whereas serine 421 (S421) is dephosphorylated in resting neurons but phosphorylated in active neurons. Two in vitro studies have shown that neuronal activity- induced phosphorylation at S421 precedes the release of MeCP2 from the neuronal specific promoter of the brain-derived neurotrophic factor (BDNF) gene and the subsequent expression of BDNF. Collectively, those studies raise the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, leading to important consequences in development and function of the adult brain. To test this hypothesis in vivo, we have generated several novel Mecp2 knockin alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on the MeCP2 protein. As a part of our long-term goal to understand the dynamic role of MeCP2 in DNA methylation-dependent epigenetic regulation of mammalian brain development and functions, we propose to: 1) study the effects of manipulating MeCP2 phosphorylation on animal behavior; 2) study the effects of manipulating MeCP2 phosphorylation on adult neurogenesis; 3) study how MeCP2 phosphorylation regulates its binding to the Bdnf promoter, remodels chromatin and subsequently alters BDNF expression and neuronal activity. Together, the experiments proposed in these three specific aims will provide insights into the central role of neuronal activity induced differential phosphorylation of MeCP2 in regulating neuronal gene expression, and its functional significance in neuronal development and animal behavior. These insights will not only bring us closer to understand the molecular mechanism of RTT and find potential treatments for RTT, but also benefit the general understanding of autism. PUBLIC HEALTH RELEVANCE: Results from this study will not only help elucidate the central role of MeCP2 in DNA methylation-dependent epigenetic regulation of brain function, but also advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because the considerable overlap in clinical features between RTT and autistic spectrum disorders, the lessons learned studying RTT might also benefit the general understanding of autism.